JOURNAL OF VIROLOGY, Apr. 1979, p. 339-350 0022-538X/79/04-0339/12$02.00/0

Vol. 30, No. 1

Biochemical Studies on the Phlebotomus Fever Group Viruses (Bunyaviridae Family) GLORIA ROBESON,' LAILA H. EL SAID,' WALTER BRANDT,' JOEL DALRYMPLE 2 DAVID H. L. BISHOP*

AND

Department of Microbiology, The Medical Center, University of Alabama in Birmingham, Birmingham, Alabama 35294,' and Department of Virus Diseases, Walter Reed Army Institute of Research, Washington, D.C. 200122

Received for publication 15 November 1978

Analyses of the virion polypeptides and genomes of several Phlebotomus fever viruses, Karimabad, Punta Toro, Chagres, and the sandfly fever Sicilian serotype viruses, have estblished that they are biochemically similar to the accepted members of the Bunyaviridae family. Like snowshoe hare virus (a member of the California serogroup of the Bunyavirus genus of the Bunyaviridae family), Karimabad, Punta Toro, Chagres, and the sandfly fever Sicilian serotype viruses all have three viral RNA species, designated large (L), medium (M), and small (S). Oligonucleotide fingerprint analyses of Karimabad and Punta Toro virus RNA species indicated that their L, M, and S RNA species are unique. By polyacrylamide gel electrophoresis it was determined for Karimabad virus that the apparent molecular weights of its L, M, and S RNA species are 2.6 x 10", 2.2 x 106 and 0.8 x 106, respectively. For Punta Toro virus, the apparent molecular weights of its L, M, and S RNA species are 2.8 x 10", 1.8 x 10", and 0.75 x 10", respectively. The major internal nucleocapsid (N) protein of Karimabad virus was found to have a molecular weight of 21 x 103. A similar polypeptide size class was identified in preparations of sandfly fever Sicilian serotype, Chagres, and Punta Toro viruses. The Karimabad virus glycoproteins formed the external surface projections on virus particles and could be removed from virus preparations by protease treatment. The glycoproteins in an unreduced sample could be resolved into two size classes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They had apparent molecular weights of 62 x 103 and 50 x 10:' in continuous polyacrylamide gels. When Karimabad virus preparations were reduced with 1% f3-mercaptoethanol, prior to resolution by continuous polyacrylamide gel electrophoresis, all the viral glycoprotein was recovered in a single size class, having an apparent molecular weight of 62 x 10'. Two or three major virion polypeptides have been identified in preparations of Punta Toro, Chagres, and sandfly fever Sicilian serotype viruses. group

,

The Bunyaviridae family of arthropod-borne viruses has 89 registered members in the genus Bunyavirus, formerly known as the "Bunyamwera supergroup of viruses" (1, 29, 30). The type virus of the genus (and the family) is Bunyamwera virus (1, 8, 9, 29, 30). Another 56 viruses, including the Phlebotomus fever (PHL) group viruses and Uukuniemi virus, are considered as possible members of the family (1, 29, 30). Many of these possible members are morphologically and morphogenically similar to the accepted members of the genus (15, 20), so that they have been termed bunyavirus-like viruses. The 11 serogroups of viruses in the Bunyavirus genus have been grouped together on the basis of serological cross-reactivities (1, 29, 30, 36). Certain, but not necessarily all, members of

each serogroup are cross-reactive with particular, but not necessarily all, members of another serogroup. This has led to the concept of the Bunyamwera supergroup of viruses (20, 29, 30). The limited molecular and genetic studies of bunyaviruses that have been published have substantiated that the viruses in the supergroup are structurally comparable to one another (7, 10-14, 17, 19, 21, 22, 24-28, 32, 33, 35; L. H. El Said et al. Am. J. Trop. Med. Hyg., in press). The principal criterion which sets these viruses apart from other families of RNA viruses is the fact that the genetic information of bunyaviruses is negative sense and is resident in three segments of RNA (5-7, 10-14, 17, 22, 24, 26-28, 31; El Said et al., in press). Other than 6 unassigned viruses, the 56 pos339

340

ROBESON El' AL.

sible members of the Bunyaviridae family have been placed into 12 serogroups (1, 29, 30). The largest serogroup is the PHL group, which has 22 members (1, 16, 34). Although morphologically the PHL group viruses are comparable to one another (20), and to other members of the family, no biochemical or genetic evidence has been published to substantiate the placement of these viruses in the Bunyaviridae family. We report in this paper structural analyses of certain PHL group viruses and show that these viruses have a segmented genome resident in three species of RNA. The virion polypeptides of PHL group viruses are comparable to those of Uukuniemi and the Bunyamwera supergroup viruses. These studies therefore support the placement of the PHL group viruses in the Bunyaviridae family. MATERIALS AND METHODS Reagents. Radioisotopes were purchased from ICN, Irvine, Calif. a-Chvmotrypsin was obtained from Sigma Chemical Co., St. Louis, Mo. Viruses and cells. Snowshoe hare, Chagres, sandflv fever Sicilian serotvpe, Punta Toro (PT), and Karimabad (KAR) viruses were obtained as mouse brain-passaged virus from R. Shope, Yale Arbovirus Research Unit, New Haven, Conn. Vero cells were obtained from R. Tesh, Pacific Research Station, Honolulu, Hawaii. Plaque assays, growth, and purification of viruses. Snowshoe hare virus was grown in BHK-21 cells in the presence of sodium 3Pi, r[ Hileucine, or 1Clabeled amino acids and was purified as described previously (10, 13, 21-23). The PHL group viruses were each plaque purified at 35°C by use of confluent monolayers of Vero cells and an overlay which consisted of medium 199 lacking phenol red (Microbiological Associates, Walkersville, Md.) mixed with 1Y. (wt/ vol) purified agar (Difco), essential vitamins and amino acids (1 x concentration, Microbiological Associates), 1%e (vol/vol) dimethyl sulfoxide (Sigma Chemical Co.), 10 U of penicillin/ml, 10 tig of streptomycin/ml, 10% (vol/vol) heat-inactivated fetal calf serum (HEM, Rockville, Md.), and 0.02c DEAE-dextran (Sigma), buffered with 0.3%; (wt/vol) sodium bicarbonate. The dimethyl sulfoxide and DEAE dextran are required for obtaining plaques with the sandfly fever viruses, but not for the other PHL group viruses used in this study (J. M. McCown, W. E. Brandt, W. H. Bancroft, and P. K. Russell, Am. J. Trop. Med. Hyg., in press). The plaque assays were overlaid 4 to 7 days later (depending on the virus strain) with 1F% (wt/wt) purified agar (Difco) made up in 0.85%i (wt/ vol) saline containing 0.133 mg of neutral red/ml. Plaques (1 to 4 mm in diameter) were counted and picked 1 day later. Working stocks of the PHL group viruses were obtained by infecting Vero cells with the mouse brainpassaged virus or the virus eluted from a plaque and growing them for 3 days at 35°C. The growth medium consisted of Eagle basal medium (Earle salts, Microbiological Associates), containing 5% (vol/vol) heat-

J. VIROI,.

inactivated fetal calf serum (HEM), 1%; (wt/vol) Lglutamine, 100 U of penicillin/ml, and 0.1 mg of streptomvcin/ml. After two and three serial passages in Vero cells, working stocks of the PHL viruses were obtained with titers of 2.2 x 10' PFU/ml (Sicilian sandflv fever), 2.1 x 10 I'FU/ml (Chagres). 6.5 x 10 PFU/ml (KAR), and 5.3 x 10' PFU/ml (PT). Virus stocks were kept at -70°C in the presence of 20'% (vol/ vol) heat-inactivated fetal calf serum. Labeled virus preparations were obtained by growing the IPHL group viruses for 3 days at 350C in growth medium containing [rHlleucine (10 jiCi/ml), '4C-labeled amino acid mixture (1 [Ci/ml), [rHiuridine (20 1Ci/ml), sodium 32Pi (200 1Ci/ml), or [LH]glucosamine (5 iCi/ml). The viruses were purified by polyethylene glycol-NaCl precipitation followed bv two 90-min cycles of glycerol-potassium tartrate gradient centrifugation as described previously (21, 23). After thev were harvested from the second gradient, the viruses were dialyzed overnight against either 0.01 M Tris-hvdrochloride buffer, pH 7.4, or 0.15 M NaCl, in 0.01 M T'ris-hvdrochloride buffer, pH 7.4. In initial experiments virus preparations were purified by successive glycerol-potassium tartrate and sucrose gradient centrifugation followed by pelleting (21, 23). Since this latter procedure proved deleterious for obtaining sufficient yields of intact virus preparations, the former procedure was subsequently adopted (see Discussion). Polyacrylamide gel electrophoresis for resolving viral polypeptides. Virus preparations in 0.01 M Tris buffer were dissociated by 1%. (wt/vol) sodium dodecyl sulfate (SDS), 1 M urea, and 0.01 M sodium phosphate, pH 7.0, and were incubated in the presence or absence of 15. (vol/vol) /3-mercaptoethanol at 60°C for 30 min. The viral polypeptides were resolved by electrophoresis in continuous 8%4 (wt/vol) polyacrylamide gels containing 0.1 M sodium phosphate buffer, pH 7.0, 0.1'%c SDS, and 6 M urea as described previously (23). The distribution of radioactivity was determined after slicing each gel into 1-mm sections and eluting the radioactivity in Protosol (New England Nuclear Corp., Boston, Mass.) for 18 h at 37°C before addition of a toluene-based scintillation cocktail. T'he resolution of viral polypeptides by discontinuous polyacrylamide slab gel electrophoresis was performed as described elsewhere (13, 21, 23). After electrophoresis, gels were fluorographed by the procedure of Bonner and Laskey (4) by treatment first with dimethyl sulfoxide, then dimethyl sulfoxide containing 2,5-diphenyloxazole, and finally water. The gels were dried and autoradiographed. Autoradiographs were scanned at 550 nm in a Schoeffel double-beam spectrodensitometer (Schoeffel Instrument Corp., Westwood, N.J.). Polyacrylamide gel electrophoresis for resolving RNA species. Labeled virus or cell preparations were extracted for RNA, and the RNA species were resolved by electrophoresis in 2.4%. gels of polyacrylamide as described previously (2, 13, 22). Resolution of the viral RNA species by SDSsucrose gradient centrifugation, RNase T, digestion of RNA, and separation of the oligonucleotides by two-dimensional gel electrophoresis. The extraction and purification of 2P-labeled bunyavirus L, M, and S RNA species have been described

VOL. 30, 1979

PHLEBOTOMUS FEVER GROUP VIRUSES

previously (10). RNA samples, dissolved in 15 [LI of 0.02 M Tris-hydrochloride-0.002 M EDTA (pH 7.4), were digested with 10 U of RNase T, at 37°C for 30 min, and the resulting oligonucleotides were resolved by two-dimensional polyacrylamide gel electrophoresis (10). After electrophoresis, each gel was autoradiographed to obtain the oligonucleotide fingerprint

(10). Electron microscopy. Samples were applied to 300-mesh copper grids with carbon-coated Formvar films and stained with 2% (wt/vol) sodium phosphotungstate, pH 6.2. Specimens were examined in a Philips 301 electron microscope. Polyethylene glycol-dextran T-500 phase separation of dissociated virus components. The procedures used to phase-separate viral nucleocapsids from solubilized virion components after treatment with Triton X-100 have been described (3).

RESULTS

Growth capabilities of KAR, PT, Chagres, and Sicilian sandfly fever viruses in Vero cells. When confluent monolayers of Vero cells were infected with KAR virus at multiplicities of infection of 0.1 to 20 PFU per cell, the maximum yields of infectious progeny virus (1 x 10' to 5 x 10' PFU/ml) were obtained 2 to 3 days postinfection for cells incubated at 35 to 38°C.

341

For infections initiated at lower multiplicities of infection (e.g., 0.001 PFU/cell), slightly higher yields of infectious virus were obtained (1 x 108 to 2 x 108 PFU/ml), with maximum titers achieved by 5 days postinfection. Essentially similar results were obtained for Chagres, PT, and Sicilian sandfly fever infections. Viral RNA species of Sicilian sandfly fever, Chagres, KAR and PT viruses. Central to the question of whether the PHL group viruses are like other members of the Bunyaviridae family is whether they have genomes consisting of three segments of RNA. The presence of a segmented genome for Sicilian sandfly fever, Chagres, KAR, and PT viruses was investigated by analyzing their 12P-labeled viral RNA species by SDS-sucrose gradient centrifugation (Fig. 1). For all four viruses, three peaks of radioactivity were obtained which, in view of their sizes, have been designated large (L), medium (M), and small (S). The sedimentation coefficients of the three RNA species for all four viruses were estimated by comparison to marker BHK-21 28S and 18S rRNA species, run in a parallel gradient, giving values of 29 ± 2S, 24 + 2S, and 18 + 2S, for L, M, and S, respectively. To further characterize the viral RNA species

Punta Toro M

L

Chogrese

S

8 32 p

Cpm x

10i3